Fungal attacks to root systems and crowns of declining Fraxinus exelsior

1 - 06 71

Fungal attacks to root systems and crowns of declining Fraxinus excelsior

Remigijus Bakys¹, Rimvis Vasiliauskas², Pia Barklund², Katarina Ihrmark² and Jan Stenlid²

1Deptartment of Plant Protection, Lithuanian University of Agriculture, LT-4324 Kaunas, Lithuania

2Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden [email protected]

Abstract

The aim of this study was twofold: 1) to investigate the extent of decay in roots and stems of declining ash; 2) to determine fungal species in damaged roots and shoots, and estimate their potential pathogenicity. In central Lithuania, 33 ash trees showing various degree of decline were felled and their root systems excavated. The positive correlation was detected between severity of the dieback and amount of decayed roots, length of decay within the stems and extent of decay over stump cross-section. A total of 150 isolations from root systems (3 samples from 50 root systems: at 0.5 m, 1 m and 1.5 m away from a stem) yiel- ded 96 isolates representing 28 fungal species. Another 195 fungal isolates with 36 identified species were obtained from sound looking, damaged and heavily dama- ged shoots. Armillaria cepistipes was the fungus, most fre- quently isolated from root samples, whereas Giberella avenacea, Alternaria alternata and Epicoccum nigrum dominated among crown infecting species. Subsequently, 27 fungal species isolated from decayed roots and 18 spe- cies from shoots were tested for pathogenicity against 600 one year-old Fraxinus excelsior seedlings.

Introduction

The issue of declining European ash (Fraxinus excelsior L.) became important since mid-1990s, when this process was initially observed in Poland and Lithuania. Subsequ- ently conducted studies did not reveal any correlation between tree mortality and geographic location of a stand, forest site type, age of a stand, species composition and edaphic factors (Juodvalkis & Vasiliauskas 2002, Przybyl 2002, Lygis et al. 2005). Characteristic symptoms of the disease are gradual crown decline due to necrotic patches on shoots and stems.

However, there were certain differences in pathological process of ash decline in different geographic areas. From Lithuania, for example, heavy root and butt rot of dying and dead trees was reported, cause of which was Armilla- ria cepistipes Velen. (Lygis et al. 2005). By contrast, in other countries damage to shoots and branches is thought to be of crucial importance for the decline, and no decay of stem bases and roots was observed (Przybyl 2002, Bark- lund 2005). In order to acquire more knowledge about pathological process in different parts of a tree, during the present study we investigated: 1) the extent of decay in roots and stems of declining ash and its correlation with the severity of the dieback; 2) fungi that invade roots and shoots of diseased trees and their relative pathogenicity.

Materials and methods

The methodology of this study consists of three basic parts:

examination and fungal isolation from root systems and crowns, and pathogenicity tests with the isolated fungi.

Root systems were investigated in three 50–100 year- old F. excelsior stands located in south western part of Lit- huania, Sakiai forestry district. The trees were of four health categories: 1) slight crown damage (dieback of up to 25 % of shoots); 2) moderate crown damage (up to 50 %);

3) severe damage (up to 75 %); 4) crown death (100 %). A total of 33 trees from all four categories were chosen for further investigation. They were situated at least 20 m from each other. The trees were cut down and the extent of decay in stump, stem base and roots (longitudinal and over cross-section) was estimated. For this, the root systems of cut trees were excavated about 40 cm deep at 1m radius from a stem base. Also, the percentage of decayed roots thicker than 2 cm was calculated. For fungal isolations, 150 wood pieces were taken from roots of 50 moderately damaged trees, – one root per tree, 3 wood samples per root (at 0.5 m, 1 m and 1.5 m distance from stem respectively).

Crowns of declining F. excelsior were examined in two sites in Sweden, one near Örebro (central Sweden), and another one near Visby (Gotland). The trees with crown dieback symptoms were cut and branch samples were taken. Depending on symptoms at the shoot base, all shoots were divided in three health categories: sound loo- king, with initial necroses at the shoot base and with advanced necroses. From the shoot bases, altogether 171 wood samples (58 from first, 58 from second and 55 from third health group, respectively) were taken for fungal iso- lations.

Pure cultures of fungi were isolated from about 4 x 0.5 cm wood pieces taken from roots, and 2 x 0.5 cm pieces of wood and bark taken from shoots. The pieces were cut out, sterilized in open fire and plated on Petri dishes containing Hagem agar. All samples were incubated at room tempera- ture for two weeks. All obtained fungal pure cultures were grouped depending on mycelial morphology. The repre- sentatives of each groups, were selected for molecular identification by ITS sequencing (White et al. 1990), similarly as in our previous study (Vasiliauskas et al.

2005). Sequence results were checked against available databases – NCBI BLAST database (Altshul et al. 1997), and database of the Dept. of Forest Mycology and Path- ology at the Swedish University of Agricultural Sciences.

A total of 27 fungal species, isolated from decayed roots and 18 species, isolated from shoots were tested for pathogenicity against 600 one year-old F. excelsior seed- lings planted under bare root conditions. Pieces of wood 1

u

u

Aktuelt fra skogforskningen 72

respective strain, were used as an inocula. Sterile wood pieces were used as control. They were attached with a tape to a 1

u

Results and discussion

The amount of decayed roots varied from 10 to 30 % in trees with slight crown damage, from 20 to 70 % in trees with moderate crown damage, from 30 to 90 % in trees with severe crown damage, and from 80 to 100 % in dead trees. The corresponding values for length of decay in a butt of a stem were 0.1–0.4 m, 0.2–1.5 m, 0.4–1.6 m and 0.4–2.5 m. For extent of decay over stem cross-section the corresponding values for the health categories were 10–

20 %, 5–60 %, 30–60 %, and 70–100 %. As a result, there were positive correlations between severity of the dieback and amount of decayed roots (rS = 0.86), length of decay in a butt of a stem (r_S= 0.57), and extent of decay over stump cross-section (r_S = 0.87).

The isolations from roots yielded 96 fungal strains representing 24 species. Mainly the same species of fungi were isolated from roots at different distances from the stem (0.5, 1 and 1.5 m), as in comparisons between the communities Sorensen indices of quantitative similarity (Magurran 1988) were high (S_N = 0.84–0.96). However, general species richness was relatively high and species accumulation curve was not asymptotic, indicating that increased sampling effort in obtained roots would reveal additional species of fungi.

The dominating basidiomycete was Armillaria spp. In addition, some other wood-decomposing basidiomycetes, as Coprinus disseminatus and Pholiota carbonaria were also present. Characteristic ascomycetes were Nectria spp., Xylaria sp. and Scytalidium lignicola. Although mating tests with the isolates of Armillaria spp. were not performed in the present study, we suspect species to be A.

cepistipes, as this species was reported to invade stem bases of declining F. excelsior in other parts of Lithuania (Lygiset al. 2005). On the other hand, the cited study also demonstrated that the fungus is not the primary cause of F.

excelsior decline, as its genotypes on examined sites was large and several decades old, when the decline there has been recorded only few years previously (Lygis et al.

2005). Moreover, A. cepistipes is known as weak oppor- tunistic pathogen, invading trees under stress, weakened by some other factor (Entry et al. 1986). Moreover, during earlier extensive field observations sporocarps of the fungus on Fraxinus had not been observed (Sokolov 1964), indicating that this tree species is somehow unusual host.

The isolations from shoot bases yielded 195 fungal strains representing 36 species. Mainly the same species of fungi were isolated from crown samples collected at differ- ent localities (Örebrö and Visby), as in comparisons between the communities Sorensen indice of quantitative similarity (Magurran 1988) was high (S_N = 0.89). How- ever, general species richness was relatively high and spe-

cies accumulation curves from both localities were not asymptotic, indicating that increased sampling effort in crowns would reveal additional species of fungi.

Species most commonly isolated were asco- and deute- romycetes: Alternaria alternata,Fusarium spp., Epicco- cum nigrum,Lewia sp., Botryosphaeria stevensii,Phom- opsis sp., Phoma glomerata Cladosporium sp., Cytospora spp. and many others. Occasionally, in shoots we recorded the presence of wood decay basidiomycetes – Coprinus sp., Pharenochaete spp., and one unidentified basidiomy- cete. As in our work, many similar or related asco- and deuteromycetes were detected in crowns and stems of decliningF. excelsior during the recent studies in Poland and Lithuania (Przybyl 2002; Lygis et al. 2005; Kowalski

& Lukomska 2005). However, the question of which of those are primarily responsible for the dieback of crowns, to date remains largely unclear, and we look forward towards the evaluation of the pathogenicity tests.

References

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W

& Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402.

Barklund P 2005. Askdöd grasserar över Syd- och Mellansverige. (In Swedish). SkogsEko no.3: 1, 11–13.

Entry J A, Martin NE, Cromack K Jr & Stafford SG 1986. Light and nutrient limitation in Pinus monticola: seedling susceptibility to Armillaria infection. For Ecol Manage 17: 189–198.

Juodvalkis A & Vasiliauskas A 2002. Lietuvos uosynu ^{dži vimo} apimtys ir jas lemiantys veiksniai. (In Lithuanian with English summary: The extent and possible causes of dieback of ash stands in Lithuania). LŽ U Mokslo Darbai, Biomedicinos Mokslai 56: 17–22.

Lygis V, Vasiliauskas R, Larsson K & Stenlid J 2005. Wood-inhabi- ting fungi in stems of Fraxinus excelsior in declining ash stands of northern Lithuania, with particular reference to Armillaria ce- pistipes. Scand J For Res 20: 337–346.

Kowalski T & ukomska A 2005. The studies on ash dying (Fraxinus excelsior L.) in the Woszczowa Forest Unit stands. Acta Agrobot 59: 429–440.

Magurran AM 1988. Ecological Diversity and Its Measurement.

Princeton University Press, Princeton, 197 pp.

Przybyl K 2002. Fungi associated with necrotic apical parts of Fraxi- nus excelsior shoots. For Path 32: 387–394.

Sokolov DV 1964. Kornevaya Gnil’ ot Openka i Bor’ba s nei. [Ar- millaria Root Rot and Its Control.] (In Russian.), 182 pp.

Lesnaya Promyshlennost’, Moscow.

Vasiliauskas R, Larsson E, Larsson K-H & Stenlid J 2005. Persisten- ce and long-term impact of Rotstop biological control agent on mycodiversity in Picea abies stumps. Biol Contr 32: 295–304.

White TJ, Bruns T, Lee S & Taylor J 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenethics.

In Innis MA, Gelfand DH, Sninsky JJ & White TJ (eds). PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc. San Diego, CA, pp 315–322.

u

U